Lecture 25

Review

Medical Optics and Lasers

• Application of optical methods to medicine

• Why optical methods?– Non-invasive– No side-effects– High resolution– Functional information– Real-time information– Cost effective– Portable

Medical Optics and Lasers

• Optical methods based on interactions of light with matter (biological sample)– Basic Principles– Absorption– Scattering

• Multiple scattering/Diffusion• Single scattering

– Fluorescence– Microscopy– Optical Coherence Tomography– Photodynamic therapy

Light as a wave

Period

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)/(1

)(sec

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frequencyangular

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meterswavelength

Monochromatic (only onewavelength/frequency)waves traveling in phase

Monochromatic (only onewavelength/frequency)waves traveling out of phase

Phase==t-kz

kzttz o cos,

Matter: Basic principles

• The basic unit of matter is the atom• Atoms consist of a nucleus surrounded by

electron(s)• It is impossible to know exactly both the

location and velocity of a particle at the same time

• Describe the probability of finding a particle within a given space in terms of a wave function,

Particle in a box

• The particle confined in a one-dimensional box of length a, represents a simple case, with well-defined wavefunctions and corresponding energy levels

• n can be any positive integer, 1,2,3…, and represents the number of nodes (places where the wavefunction is zero)

• Only discrete energy levels are available to the particle in a box----energy is quantized

a

xn

axn

sin2

)( 2

22

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hnEn

Atomic orbitals: Quantum numbers

•Principal quantum number, n–Has integral values of 1,2,3…… and is related to size and energy of the orbital

•Angular quantum number, l–Can have values of 0 to n-1 for each value of n and relates to the angular momentum of the electron in an orbital; it defines the shape of the orbital

•Magnetic quantum number, ml

–Can have integral values between l and - l, including zero and relates to the orientation in space of the angular momentum.

•Electron spin quantum number, ms –This quantum number only has two values: ½ and –½ and relates to spin orientation

Molecular orbitals• Molecular orbitals (chemical bonds) originate from the overlap of

occupied atomic orbitals

• Bonding molecular orbitals – are lower in energy than corresponding atomic orbitals (stabilizes the

molecule)

• Anti-bonding orbitals – are higher in energy than corresponding atomic orbitals and destabilizes

the molecule

bonds – involve overlapping s and p orbitals along the line joining the nuclei of

the bond-forming atoms

bonds – involve p and d orbitals overlapping above and below the line joining the

nuclei of the bond-forming atoms

Hybrid orbitals and conjugated bonds

• The four 2p orbitals can combine to form these orbitals, arranged according to energy, with the lowest energy orbital at the bottom.

• Can you think of a set of wavefunctions that may describe what is going on?

• These are similar to the wavefunctions we got for a particle in the box, with the length of the box corresponding to the length of the carbon chain

Principles of laser operation

• Stimulated emission• Population inversion• Laser cavity

– Main components– Gain and logarithmic losses– Two vs. three vs. four-level systems– Properties of laser light– Homojunction/heterojunction semiconductor

lasers

Cell and Tissue basics

• Basic components of a cell– Nucleus– Mitochondria– Lysosomes– ER – Golgi

• Basic components of epithelial tissues– Types of epithelia– Connective tissue– Basement membrane

Light-tissue interactions

• scattering– elastic scattering

• multiple scattering

• absorption

• fluorescence

Epithelium

Connective Tissue

• single scattering

Optical methods are based on different types of light-matter interactions to provide structural, biochemical, physiological and morphological information

Tissue optical properties

• There are two main tissue optical properties which characterize light-tissue interactions and determine therapeutic or diagnostic outcome:– Absorption coefficient: a (cm-1)

a=a*Na =(A/L)*ln10 a=atomic absorption cross section (cm2)• Na=# of absorbing molecules/unit volume (cm-3)• A=Absorbance• L=sample length

– Scattering coefficient: s (cm-1) s=s*Ns s=atomic scattering cross section (cm2)• Ns=# of scattering molecules/unit volume (cm-3)

Tissue absorption

Major tissue absorbers include: Hemoglobin, lipids (beta carotene), melanin, water, proteinsOxy and deoxy hemoglobin have distinct spectra. Optical measurements can provide information on tissue oxygenation, oxygen consumption, blood hemodynamics

Tissue scattering spectra exhibit a weak wavelength dependence

Structural proteins constitute major tissue scattering centers. Cell nuclei and membrane rich organelles (e.g. mitochondria) also scatter light

Fluorescence spectra provide a rich source of information on

tissue state

-1

-0.5

0

0.5

1

1.5

350 400 450 500 550 600300

350

400

450

Emission (nm)

Exc

itatio

n (

nm)

NADH

FAD

Collagen

Trp

Protein expression

Structural integrity

Metabolic activity

Courtesy of Nimmi Ramanujam, University of Wisconsin, Madison

Which optical method to use?

• Three main questions:– What is the required depth of penetration?– What is the acceptable resolution?– What type of information is needed?

1 mm 1 cm 10 cm

Penetration depth (log)

1 m

10 m

100 m

1 mm

Resolution (log)

OCT

Imaging methods

100 nm

100 m10 m1 m

Standardmicrosc

4-Pi/STED

Confocal/multi-photon microscopy

Diffuse optical tomography and spectroscopy

Spectroscopic methods: Functional information• Diffuse reflectance

– Penetration depth: microns to centimeters depending on wavelength, souce/detector separation, light delivery/collection geometry

– Resolution not well defined– Absorption

• Tissue oxygen saturation• Arterial/venus oxygen saturation• Oxygen consumption• Hemodynamics

– Scattering• Structural changes of the matrix• May be nuclear changes

• Light Scattering– Penetration depth: microns to hundreds of microns depending on how highly scattering is the sample– Inelastic scattering (Raman)

• Information: biochemical composition– Elastic scattering

• Information– Size distribution of major cell scattering centers (e.g. nuclei, mitochondria)– Cell/tissue organization

• Resolution– Potential to detect size changes on the order of 100 nanometers

• Fluorescence– Penetration depth: microns to centimeters depending on implementation, i.e. wavelength, sample optical

properties, source/detector geometry– Endogenous fluorescence

• Cell and tissue biochemistry (NADH/FAD, tryptophan, porphyrins, oxidized lipids• Tissue structure (collagen, elastin)

– Induced fluorescent protein expression (molecular specificity)– Fluorescent tags

• Antibodies (antigen expression)• Molecular beacons (enzyme activity)

Diffuse optical tomography and spectroscopy

• Applications– Breast cancer detection– Brain function– Oxygen consumption by muscles– Arthritis– atherosclerosis– Pulse oximeter– Jauntice (billirubin) test for neonates

Light scattering spectroscopy

• Cancer detection

• Detection of pre-cancerous changes– Barrett’s esophagus– Uterine cervix– Oral cancers

• Biopsy guidance

• Non-invasive patient monitoring

Optical coherence tomography

• Non-invasive detection of morphological changes

• Applications– Cancer detection– Eye diseases– Atherosclerosis– Developmental biology

Raman scattering

• Applications– Atherosclerosis– Cancer detection– Blood composition– Bacterial detection

Tissue fluorescence

• Applications– Cancer detection

• Pre-cancer detection• Guide to biopsy• Patient monitoring

– Atherosclerosis detection– Bacterial infection (?)

Microscopy

• Cell microscopy– Understand basic cell functions in healthy and

disease states– Understand role of specific proteins and cell

component interactions

• Tissue/intravital microscopy– Understand cell matrix interactions that govern

disease development, progression and regression

• Drug/therapy development and optimization• Early detection

Multi-modality optical detection

• Goal: Acquire morphological and biochemical information to achieve more sensitive/specific detection

• Combined use of fluorescence, diffuse reflectance and light scattering

• Combined use of Raman and fluorescence• Combined use of OCT and fluorescence• Combined use of reflectance and

fluorescence imaging

Photodynamic therapy

• Example of light-based therapeutic method

• Light used to achieve cytotoxicity

• Optical methods can also be used to tailor dosimetry to patient and monitor/predict therapeutic efficacy

• Used for treating a variety of conditions from cancer to acne to atherosclerosis

Optical methods are a powerful tool for understanding human health and improving

disease detection and treatment

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y (cm)

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